Mounting evidence suggests that epigenetic regulation is critical to diverse biological processes and diseases. Post-translational modifications (PTM) in histones are generally considered to be a major group of epigenetic marks. Many histone PTMs have been shown to contribute to the "histone language" and epigenetic program that dictates diverse DNA-templated biological outputs. Nevertheless, it remains unclear whether additional histone marks exist in cells and, if so, what are the roles of these undiscovered signals in epigenetic phenomena. We hypothesize that unidentified PTMs that regulate epigenetic mechanisms are present in mammalian histones. Our hypothesis is based on our recent identification of two novel histone modifications, propionylation and butyrylation of lysine (KProp and KButy) and other unknown PTMs in histones, the chemical structures of which remain to be defined. We therefore propose to comprehensively screen for novel histone PTMs, and subsequently define and verify their structures by a novel approach, involving analytical chemistry, organic synthesis, biochemistry/immunochemistry, and a novel bioinformatics. This integrated approach allows for comprehensive, unbiased, and sensitive identification of histone PTMs. We have demonstrated the feasibility of major components of this novel method. To our knowledge, such a systematic approach to the discovery of new histone PTMs has not been described before. We expect to identify several novel histone PTMs as a result of this study, expanding our current knowledge of histone marks. Identification of novel histone PTMs will provide a stepping stone to the research community toward investigation of the possible roles of these histone PTMs in epigenetic mechanisms and diseases. Four specific aims are proposed in this application. First, we will investigate potential epigenetic roles of histone KProp and KButy by studying their genomic distribution and dynamics in mouse embryonic stem (mES) cells. We will use a proteomics approach to identify all the residues that bear the both PTMs in the core histones from undifferentiated mESs and their differentiated cells. We will examine the possible roles of the two PTMs in epigenetic regulation by studying the dynamics of the PTMs during mES cell differentiation and their contributions to the gene expression by ChIP- qPCR and ChIP-on-chip. Second, we will identify and purify histone peptides bearing novel PTMs using an integrated approach involving liquid chromatography, HPLC/MS/MS analysis, and a novel bioinformatics algorithm. The peptide bearing a novel PTM will be isolated for structure determination. Third, we will determine the chemical structures of the novel PTM moieties by high-resolution mass spectrometric analysis and NMR spectroscopy. The novel PTM structures will be verified by various analytical techniques and Western blotting analysis. Finally, we will study dynamics and potential epigenetic roles of the novel histone PTMs in mES cells that will be identified in Aim 2 and 3, using the experiments as described in Aim 1. PUBLIC HEALTH RELEVANCE: Histone post-translational modifications (PTMs) are known to be involved in the epigenetic regulation of diverse cellular processes and diseases. The proposed study aims to identify and validate novel histone PTMs, and to study their possible roles in mouse stem cell differentiation. The resulting knowledge will expand our understanding on histone PTMs and will stimulate the studies on the epigenetic roles of the novel histone PTMs in disease progression.